Cell Envelope Signaling in Escherichia coli

The ferrichrome-iron receptor ofEscherichia coli is FhuA, an outer membrane protein that is dependent upon the energy-coupling protein TonB to enable active transport of specific hydroxamate siderophores, infection by certain phages, and cell killing by the protein antibiotics colicin M and microcin 25. In vivo cross-linking studies were performed to establish at the biochemical level the interaction between FhuA and TonB. In an E. coli strain in which both proteins were expressed from the chromosome, a high molecular mass complex was detected when the ferrichrome homologue ferricrocin was added immediately prior to addition of cross-linker. The complex included both proteins; it was absent from strains of E. coli that were devoid of either FhuA or TonB, and it was detected with anti-FhuA and anti-TonB monoclonal antibodies. These results indicate that,in vivo, the binding of ferricrocin to FhuA enhances complex formation between the receptor and TonB. An in vitro system was established with which to examine the FhuA-TonB interaction. Incubation of TonB with histidine-tagged FhuA followed by addition of Ni2+-nitrilotriacetate-agarose led to the specific recovery of both TonB and FhuA. Addition of ferricrocin or colicin M to FhuA in this system greatly increased the coupling between FhuA and TonB. Conversely, a monoclonal antibody that binds near the N terminus of FhuA reduced the retention of TonB by histidine-tagged FhuA. These studies demonstrate the significance of ligand binding at the external surface of the cell to mediate signal transduction across the outer membrane. The ferrichrome-iron receptor ofEscherichia coli is FhuA, an outer membrane protein that is dependent upon the energy-coupling protein TonB to enable active transport of specific hydroxamate siderophores, infection by certain phages, and cell killing by the protein antibiotics colicin M and microcin 25. In vivo cross-linking studies were performed to establish at the biochemical level the interaction between FhuA and TonB. In an E. coli strain in which both proteins were expressed from the chromosome, a high molecular mass complex was detected when the ferrichrome homologue ferricrocin was added immediately prior to addition of cross-linker. The complex included both proteins; it was absent from strains of E. coli that were devoid of either FhuA or TonB, and it was detected with anti-FhuA and anti-TonB monoclonal antibodies. These results indicate that,in vivo, the binding of ferricrocin to FhuA enhances complex formation between the receptor and TonB. An in vitro system was established with which to examine the FhuA-TonB interaction. Incubation of TonB with histidine-tagged FhuA followed by addition of Ni2+-nitrilotriacetate-agarose led to the specific recovery of both TonB and FhuA. Addition of ferricrocin or colicin M to FhuA in this system greatly increased the coupling between FhuA and TonB. Conversely, a monoclonal antibody that binds near the N terminus of FhuA reduced the retention of TonB by histidine-tagged FhuA. These studies demonstrate the significance of ligand binding at the external surface of the cell to mediate signal transduction across the outer membrane. High affinity iron uptake in Gram-negative bacteria such asEscherichia coli is a stepwise process that involves recognition of a ferric iron chelator (siderophore) by a receptor within the outer membrane, translocation of the siderophore-Fe(III) complex into the periplasm, and internalization of the iron by a cytoplasmic membrane permease in a periplasmic binding protein-dependent manner (reviewed in Refs. 1Postle K. Mol. Microbiol. 1990; 4: 2019-2025Crossref PubMed Scopus (146) Google Scholar, 2Postle K. J. Bioenerg. Biomembr. 1993; 6: 591-601Google Scholar, 3Braun V. FEMS Microbiol. 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Bacteriol. 1993; 175: 3146-3150Crossref PubMed Scopus (147) Google Scholar), transport of which shares common elements with siderophore-Fe(III) transport mechanisms, identified the importance of the TonB and ExbB/D proteins of the cytoplasmic membrane to couple proton-motive force with active transport at the outer membrane (Ref.10Larsen R.A. Thomas M.G. Wood G.E. Postle K. Mol. Microbiol. 1994; 13: 627-640Crossref PubMed Scopus (85) Google Scholar; reviewed in Refs. 2Postle K. J. Bioenerg. Biomembr. 1993; 6: 591-601Google Scholar and 11Kadner R.J. Mol. Microbiol. 1990; 4: 2027-2033Crossref PubMed Scopus (181) Google Scholar). TonB homologues have been identified in many Gram-negative bacteria, including Salmonella enterica serovar Typhimurium (12Hannavy K. Barr G.C. Dorman C.J. Adamson J. Mazengera L.R. Gallagher M.P. Evans J.S. Levine B.A. Trayer I.P. Higgins C.F. J. Mol. Biol. 1990; 216: 897-910Crossref PubMed Scopus (100) Google Scholar), Yersinia enterocolitica (13Koebnik R. Bäumler A.J. Heesemann J. Braun V. Hantke K. Mol. Gen. Genet. 1993; 237: 152-160Crossref PubMed Scopus (38) Google Scholar), Haemophilus influenzae (14Jarosik G.P. Sanders J.D. Cope L.D. Muller-Eberhard U. Hansen E.J. Infect. Immun. 1994; 62: 2470-2477Crossref PubMed Google Scholar), andPseudomonas aeruginosa (15Poole K. Zhao Q. Neshat S. Heinrichs D.E. Dean C.R. Microbiology. 1996; 142: 1449-1458Crossref PubMed Scopus (59) Google Scholar). In addition, complexes between TonB or its homologues and other proteins (ExbB, ExbD, and as yet unidentified proteins) were detected with anti-E. coli TonB monoclonal antibodies (mAbs 1The abbreviations used are: mAb, monoclonal antibody; PVDF, polyvinylidene difluoride; NTA, nitrilotriacetate; LDAO, N,N-dimethyldodecylamine N-oxide; PAGE, polyacrylamide gel electrophoresis; TLN, Tris-HCl/LDAO/NaCl. ; Ref. 16Larsen R.A. Myers P.S. Skare J.T. Seachord C.L. Darveau R.P. Postle K. J. Bacteriol. 1996; 178: 1363-1373Crossref PubMed Google Scholar). These findings demonstrate that the TonB-dependent energy transduction system is shared among many Gram-negative bacteria and suggest that high affinity energy-dependent iron uptake in Gram-negative aerobes is accomplished using TonB and its accessory proteins (16Larsen R.A. Myers P.S. Skare J.T. Seachord C.L. Darveau R.P. Postle K. J. Bacteriol. 1996; 178: 1363-1373Crossref PubMed Google Scholar). Physical association between TonB and a TonB-dependent outer membrane receptor was first demonstrated biochemically byin vivo cross-linking experiments in which TonB was coupled to the E. coli enterobactin (also known as enterochelin) receptor, FepA (17Skare J.T. Ahmer B.M.M. Seachord C.L. Darveau R.P. Postle K. J. Biol. Chem. 1993; 268: 16302-16308Abstract Full Text PDF PubMed Google Scholar). This finding substantiated genetic analyses that suggested interactions between TonB and other outer membrane transporters, namely the ferrichrome-iron receptor FhuA (18Schöffler H. Braun V. Mol. Gen. Genet. 1989; 217: 378-383Crossref PubMed Scopus (147) Google Scholar, 19Günter K. Braun V. FEBS Lett. 1990; 274: 85-88Crossref PubMed Scopus (70) Google Scholar) and the vitamin B12 receptor BtuB (20Heller K. Kadner R.J. Günter K. Gene (Amst.). 1988; 64: 147-153Crossref PubMed Scopus (142) Google Scholar, 21Bell P.E. Nau C.D. Brown J.T. Konisky J. Kadner R.J. J. Bacteriol. 1990; 172: 3826-3829Crossref PubMed Google Scholar, 22Anton M. Heller K.J. Mol. Gen. Genet. 1993; 239: 371-377Crossref PubMed Scopus (11) Google Scholar). However, formation of the FepA-TonB complex appeared to be independent of the presence of ferric enterobactin, since the strains used carried mutations in the enterobactin biosynthetic genes and were negative on chrome azurol S plates used for detecting siderophore excretion (23Schwyn B. Neilands J.B. Anal. Biochem. 1987; 160: 47-56Crossref PubMed Scopus (4493) Google Scholar). 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Microbiol. 1996; PubMed Scopus Google Scholar). that such a in FhuA as a signal to TonB in vivo, that the receptor is with the of the in FhuA and to other FepA form complexes with TonB, studies of FhuA-TonB that FhuA be to TonB and that the cross-linking between FhuA and TonB is by the presence of the ligand have established an in vitro system with which to the coupling between FhuA and TonB and the of upon the interaction. results from in vivo and in vitro experiments that ligand binding to FhuA its association with TonB. and antibodies were from The was from polyvinylidene membrane was from was from from and from was from was from The E. coli strains used in this were J. Bacteriol. PubMed Google Scholar), U. S. PubMed Scopus Google Scholar), from J. D. D. J. Bacteriol. 1990; 172: PubMed Google Scholar), Ref. R.A. Thomas M.G. Wood G.E. Postle K. Mol. Microbiol. 1994; 13: 627-640Crossref PubMed Scopus (85) Google Ref. J.T. Ahmer B.M.M. Seachord C.L. Darveau R.P. Postle K. J. Biol. 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Bacteriol. 1996; 178: 1363-1373Crossref PubMed Google Scholar) for TonB protein of reduced molecular were in at and by into with at at at and at was added to a of either or were with at to an of of the hydroxamate siderophore to as was added to of at in an of was added to a of (17Skare J.T. Ahmer B.M.M. Seachord C.L. Darveau R.P. Postle K. J. Biol. Chem. 1993; 268: 16302-16308Abstract Full Text PDF PubMed Google Scholar) was to for and and to a and for cross-linking by G.E. of Scholar, M. Chem. PubMed Scopus Google Scholar). were and in with at for proteins were on polyacrylamide and to for were (17Skare J.T. Ahmer B.M.M. Seachord C.L. Darveau R.P. Postle K. J. Biol. Chem. 1993; 268: 16302-16308Abstract Full Text PDF PubMed Google Scholar). and J. Bacteriol. 1995; PubMed Google Scholar) to between and of the FhuA R. J. Bacteriol. PubMed Google anti-TonB and to and (16Larsen R.A. Myers P.S. Skare J.T. Seachord C.L. Darveau R.P. Postle K. J. Bacteriol. 1996; 178: 1363-1373Crossref PubMed Google Scholar), of the TonB K. U. S. PubMed Scopus Google Scholar). E. strains and to an of in were by in of and by a cell at The cell was at for at to and of the outer membrane H. 1994; PubMed Scopus Google Scholar). The was at for at The from for cytoplasmic membrane H. 1994; PubMed Scopus Google Scholar), was with for at in LDAO, and to a protein of were at prior to experiments the were with LDAO, and FhuA from the outer membrane of the E. coli strain was to H. J. Mol. Microbiol. 1996; PubMed Scopus Google Scholar). the a of was in and a of of in an of was used for The was with of of proteins from the of E. coli strain was the The was with of and a of to was was at an of of were with an of for and on polyacrylamide were to and with anti-FhuA and anti-TonB The antibodies were detected either with an antibody Ref. D.E. C. PubMed Scopus Google Scholar) followed by using and or with a antibody followed by by In a a of was in TLN, with a of and a of of Addition of proteins from E. coli strain and with was performed as for the the that the presence of in the was to affinity of for the the was of to the the of upon the an in vitro of was of either or in were in with a of with of cell colicin M J. Bacteriol. PubMed Google Scholar; of a of on the strain with of cell microcin R.A. J. Bacteriol. PubMed Google Scholar; of a of or with of the or for were at anti-FhuA or were added and for These between and of FhuA, J. Bacteriol. 1995; PubMed Google Scholar). was added an of protein from as In the from was with of a of either to of or to of for prior to the addition of the to the of the was to with a of was added and for The was by and with and proteins were by addition of of of the were with for and polyacrylamide proteins were to and with anti-FhuA and anti-TonB both of which of the antibodies were detected by antibodies and of and TonB were by as a and of and of the and Molecular of the by for in of FhuA, TonB were to of experiments with were results demonstrated the of a high molecular mass complex both FepA and TonB (17Skare J.T. Ahmer B.M.M. Seachord C.L. Darveau R.P. Postle K. J. Biol. Chem. 1993; 268: 16302-16308Abstract Full Text PDF PubMed Google Scholar). that a complex between FhuA and TonB be identified by using a in cross-linking The anti-TonB detected with molecular of and with the at not have been R.A. Thomas M.G. Wood G.E. Postle K. Mol. 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Chem. 1993; 268: 16302-16308Abstract Full Text PDF PubMed Google Scholar). cross-links protein were not by prior to to the polyacrylamide The of was in in which FhuA was expressed at coli strain absent from This was detected when FhuA was expressed from the in was in the strain This a FhuA-TonB establish the of the FhuA-TonB the of the siderophore ferricrocin immediately prior to cross-linking was the of the to in strain and increased the of the complex in Addition of ferricrocin did not the protein of either or that the complex FhuA, an of proteins was by with the anti-FhuA In the proteins from strain the to FhuA of of and were of the a with an molecular mass of was detected in the from that been with was upon with the anti-TonB mAb, to was when FhuA was expressed from the in the of added The formation of this complex was dependent upon both FhuA and TonB since in strains and the complex was the complex that was detected by the anti-FhuA an of as by addition of These demonstrated that a complex between FhuA and TonB was in vivo and that its increased by with FhuA was expressed from the high a of complexes appeared the FhuA in the of These to the that were the FhuA when FhuA was expressed from the in strain in and were independent of the presence of TonB since was between and have not established the of of anti-FhuA was in vivo cross-linking with E. coli strain not In the complexes the which a of This that of FhuA not for the formation of molecular mass vitro system with which to examine the interaction between FhuA and TonB and that for of its protein was to a TonB were into the the was and proteins were with experiments that of TonB were by the in the of were to this that the of TonB be for the interaction between TonB and the The TonB which the of the and which TonB upon R.A. Wood G.E. Postle K. Mol. Microbiol. 1993; PubMed Scopus (85) Google Scholar), was used for in vitro of in the binding and retention of proteins by the of led to the recovery of In the of on the and with an of in a was detected in the of by the prior of of the for FhuA by protein not that the the FhuA to the as by with an anti-TonB The of with that the proteins in this since affinity for in the of ferricrocin complex formation between FhuA and TonB as it did in vivo, was with a of ferricrocin immediately prior to the an of that was to that used and was the an increased of was of with anti-FhuA not that the of was from the of the or presence of The in by the complex the of interaction between the proteins since ferricrocin is specific for FhuA. The in vitro results were to examine the of other such as and upon the FhuA-TonB interaction. This of a vitro to the of to ligand to FhuA. this complexes between FhuA and TonB that were in were by TonB of were used to that the TonB protein with in vitro as it did in of with anti-FhuA and anti-TonB identified in TonB binding to of the of TonB and on the established and in the of TonB with ligand The addition of ferricrocin to a in the recovery of TonB and to the of TonB to the the addition of colicin M led to a of TonB, which with from the a in the of TonB, which to and which was by the Addition of or microcin in of ferricrocin led to the recovery of of TonB that were to that ligand addition of anti-FhuA J. Bacteriol. 1995; PubMed Google Scholar) been used to FhuA to interactions between receptor and its and to H. J. Mol. Microbiol. 1996; PubMed Scopus Google Scholar). binding of to the complex the association between FhuA and TonB. the that by have been J. Bacteriol. 1995; PubMed Google Scholar) to of the of FhuA, on of the FhuA-TonB interaction into of the receptor that for the interaction. Incubation of with the complex prior to addition of TonB led to a in the of TonB that was the of is in a between and of FhuA, this that within this for association between FhuA and TonB. The that this was to by the antibody to TonB from with FhuA was since the binding of and to did not the association of TonB with FhuA and Incubation of the with the complex reduced the retention of TonB to that to the TonB of the E. coli ferric receptor was M. J. Bacteriol. PubMed Google Scholar) to TonB-dependent receptor in a that the TonB of FhuA association between FhuA and TonB in this a to of FhuA was with the cell TonB for prior to the addition of the TonB to the a to of FhuA reduced the of TonB to the complex for in vitro interaction demonstrated that TonB to the FhuA as as it did to not ferricrocin and colicin M binding to increased the of TonB, which was with from the and of a FhuA-TonB complex that FepA is not the high affinity receptor with which TonB the (17Skare J.T. Ahmer B.M.M. Seachord C.L. Darveau R.P. Postle K. J. Biol. Chem. 1993; 268: 16302-16308Abstract Full Text PDF PubMed Google Scholar) and the that TonB-dependent TonB. The finding that the ferrichrome homologue ferricrocin the interaction between FhuA and TonB results of in FhuA H. J. Mol. Microbiol. 1996; PubMed Scopus Google Scholar). of the to of FhuA led to that FhuA TonB such a its for energy to ligand across the outer membrane. The of FhuA-TonB coupling that was established by in vivo cross-linking experiments and by in vitro interaction the An of FhuA TonB, by of FhuA from a or by of of to the was to the of complex This be a of the of the presence of However, in the of FhuA, to of with anti-FhuA did not the FhuA-TonB complex in the of the complex were added it was the of the of of the FhuA-TonB complex to ferricrocin is not dependent upon the presence of ligand when the receptor is The of the FepA-TonB complex of (17Skare J.T. Ahmer B.M.M. Seachord C.L. Darveau R.P. Postle K. J. Biol. Chem. 1993; 268: 16302-16308Abstract Full Text PDF PubMed Google Scholar). this complex was not on with anti-TonB was to the mutations in to a in enterobactin results indicate that enterobactin enhances complex formation between FepA and TonB. the results of Skare (17Skare J.T. Ahmer B.M.M. Seachord C.L. Darveau R.P. Postle K. J. Biol. Chem. 1993; 268: 16302-16308Abstract Full Text PDF PubMed Google Scholar), with suggest that the FepA-TonB complex was in strains to of enterobactin by the chrome azurol S were to the in FepA that cross-linking to TonB. is the of anti-FhuA with that of FhuA, that were by of is the of proteins within did not TonB since were in a strain in which the was by of a or of the with FhuA FhuA The enterobactin receptor FepA vivo since of outer membrane FepA an the of which was that of the FepA J. J.B. P.E. U. S. 1993; PubMed Scopus Google Scholar). in vivo with followed by and with an high molecular mass that were to FepA and (17Skare J.T. Ahmer B.M.M. Seachord C.L. Darveau R.P. Postle K. J. Biol. Chem. 1993; 268: 16302-16308Abstract Full Text PDF PubMed Google Scholar). In FhuA was identified to be on the of and M. M. S. M. 1996; PubMed Scopus Google Scholar). the FhuA that was used in experiments it was that the of FhuA was However, findings with an FhuA be as a of within the outer membrane. experiments indicate that FhuA in of or not to cross-linking not The of vivo and its significance in receptor and transport to be The of the that with FhuA and that to the molecular mass complexes other cell proteins that FhuA or FhuA TonB-dependent with TonB was by that siderophore the vitamin B12 receptor, and a of near N 11Kadner R.J. Mol. Microbiol. 1990; 4: 2027-2033Crossref PubMed Scopus (181) Google E. J. Braun V. J. Bacteriol. 1987; PubMed Google Scholar, and C.D. Konisky J. J. Bacteriol. 1989; PubMed Google Scholar; for a of TonB-dependent for which the Refs. K. M.L. J. Bacteriol. 1996; 178: PubMed Google Scholar). This the is to with the TonB ligand translocation across the outer membrane. of FhuA and TonB for interaction between the The TonB is a of this to a in the interaction between TonB and TonB-dependent receptors. greatly reduced the retention of TonB by the was to specific of the FhuA-TonB interaction at or near the which is by This finding the of as specific of FhuA in the of of FhuA that for association with TonB, to the which is by of FhuA a in the binding of since of at this H. J. Mol. Microbiol. 1996; PubMed Scopus Google Scholar). mutations within the TonB of and FhuA have in included the by a either or R.J. Mol. Microbiol. 1990; 4: 2027-2033Crossref PubMed Scopus (181) Google Scholar). of such the TonB of the enterobactin receptor FepA that such in the of vivo cross-linking to TonB. The did not a yet it the of FepA at R.A. D. M. Postle K. J. Bacteriol. PubMed Google Scholar). These coupled with the finding that within the TonB be of receptor R.J. Mol. 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PubMed Google Scholar). in vitro to a of the FhuA-TonB interaction in that ferricrocin binding complex The of colicin M to a to the association between FhuA and TonB in vitro to other this the that of an energy for FhuA to signal its for TonB energy coupling H. J. Mol. Microbiol. 1996; PubMed Scopus Google Scholar). findings into the of TonB-dependent transport The of and of the protein microcin to FhuA-TonB coupling be in the receptor that from by ferricrocin and colicin M. results suggest that of anti-FhuA by of FhuA not binding to FhuA a in FhuA that in across M. J. 1996; PubMed Scopus (85) Google Scholar) and both into and ferrichrome from M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). However, not that this FhuA-TonB association since is a the of the to the other in manner the of FhuA-TonB interaction as detected in The and of proteins that the for to in receptor recognition M. Heller K.J. FEMS Microbiol. Lett. 1995; PubMed Google Scholar, M. Heller K.J. 1996; PubMed Scopus Google Scholar) in the of TonB coupling with TonB-dependent receptors. studies to upon the of FhuA which its association with TonB and upon of FhuA which for the interaction with TonB. finding that the FhuA in FhuA-TonB interactions as as did for into the of the FhuA-TonB C. for genetic and for of R. with analyses and for the a of the R. and suggested the of